The present invention relates to a method for extraction of intracellular components including intracellular metabolites. The invention addresses the problem that many substances used for extracting components from cells interfere with assays or other processing steps performed on the extracted components. The invention uses cyclodextrins to neutralise the extracting substances. In one example according to the invention, the intracellular metabolite is adenosine triphosphate (ATP) which can, after neutralisation of the extractants, be assayed using a firefly luciferinluciferase reaction. In another example, the intracellular components are nucleic acids which can, after neutralisation of the extractants, be amplified or further processed in other ways.
General aspects of extraction of intracellular components
The assay of intracellular components in biological samples is often performed by enzymatic methods. Such methods require: 1) Release of the components from the cells to make the components available to enzyme systems added in the assay. 2) Inactivation of enzymes from the cells that may act on the components during preparation, storage or assay of extracts. Extraction of the intracellular components involves opening of cell walls and membranes and release of the entire metabolite pools into the surrounding medium. Within the cells the metabolite pools often have turn-over times around a few seconds due to the action of the intracellular enzymes. As soon as an extractant starts to affect membrane integrity the enzyme systems of the cell try to counteract the resulting effects. Thus considerable changes of metabolite levels may take place during an extraction which takes time. This would obviously result in completely erroneous data on intracellular metabolite levels even using the best enzymatic assays. The only way to avoid the problem is to use extractants that rapidly open up the cell membranes and simultaneously inactivate all enzymes that act on the intracellular components. Enzyme inactivation is therefore an inherent property of all reliable extractants. The presence of a cell wall protects the cell from the extractant and makes bacterial, fungal and algal cells particularly difficult to extract. Thus strong acids with chaotropic anions like trichloroacetic acid (TCA) or perchloric acid (PCA) have frequently been used for the extraction of these types of cells. Such agents are strongly enzyme inactivating and inevitably interfere with enzymatic assays unless extracts are highly diluted before the assay. Dilution of the extracts makes it difficult to assay low concentrations of metabolites.
The more rapid the turn-over rate of the intermediate metabolite the higher is the requirement for immediate inactivation of cellular enzymes at the addition of the extractant. From this point of view ATP is one of the most difficult intracellular metabolites to extract. In all cells ATP is the means by which energy is transferred from energy yielding to energy requiring reactions. Thus many ATP converting enzymes (kinases and ATPases) exist and have high activities. Even a slight damage of membrane integrity, e.g. by an extractant, results in a rapid loss of intracellular metabolites and ions. As the cell tries to compensate for these events large quantities of ATP are consumed. One object of the work leading to this invention was to develop a reliable extraction method for microbial ATP compatible with the firefly luciferase assay. The rapid turn-over of ATP and the presence of thick cell walls in microbial cells make it likely that an extraction method for microbial ATP will work also for most other intracellular metabolites in any type of cell (unless the extractant by itself degrades the metabolite). Furthermore in the firefly luciferase assay of ATP the rate of the reaction is measured, i.e. the firefly assay is an example of kinetic assays. Thus any inhibitor added during or after the extraction will affect the assay. The activity of firefly luciferase is inhibited by a wide variety of compounds including simple salts. Firefly luciferase also has a narrow pH optimum. Thus an extraction method that works with the firefly assay is likely to work with most other enzymatic assays. This is particularly true for any end-point assay for which an inhibition can be compensated simply by extending the assay time.
Extraction of DNA and RNA
The extraction of nucleic acids from biological material forms a critical first step in many molecular biology studies. The extracted DNA or RNA is required as a substrate or template for subsequent enzymatic reactions, and hence must be biologically active. Commonly, DNA from cells or tissue is used for the amplification of specific sequences by the polymerase chain reaction (PCR) or cleavage with restriction enzymes for gene cloning or identification. The purification of genomic DNA from cells or tissue for subsequent use in gene analysis experiments conventionally involves cell lysis to release all cellular components, followed by selective digestion of proteins and RNA with specific degradative enzymes. After separation from proteinaceous material and other contaminants the DNA sample is relatively pure and functionally active. The separation step is conventionally performed by extraction with organic solvents followed by precipitation of the DNA with alcohol (J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning--A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989). Methods have been described where functionally active genomic DNA can be prepared without specific removal of contaminating protein, for example by ethanol precipitation of cell lysates (H. Xu, A.M. Jevnikar and E. Rubin-Kelly, Nucleic Acids Research 18, 4943). The critical contaminant therefore appears to be the extractant used, which is conventionally a detergent. Removal of the detergent can therefore be sufficient to allow the DNA to be used for subsequent reactions. However, conventionally detergent removal still requires a separation step, with the subsequent increase in preparation time and potential reduction in yield. A homogeneous system without any separation steps would therefore have significant advantages over current methods.
Present situation with respect to extraction and assay of microbial ATP
In rapid microbiology the firefly luciferase assay of ATP is frequently used for biomass estimations. The intracellular ATP concentration is similar in all cells and the amount of ATP per cell is approximately proportional to the intracellular volume. Bacteria contain approx. 10.sup.-18 moles of ATP per cell while fungi and algae contain considerably more ATP per cell. With simple light measuring instruments and firefly luciferase reagents 10.sup.-15 moles of ATP is easily detected in a 1 ml volume. This corresponds to approx. 10.sup.3 bacterial cells. Bacterial ATP in a biological specimen can be extracted by adding an equal volume of 2.5% trichloroacetic acid. However, to avoid interference from trichloroacetic acid with the luciferase reaction a sample volume larger than 0.01 ml cannot be used in a final assay volume of 1 ml. Thus the detection limit in the biological specimen is 10.sup.5 cells/mi. Neutralisation of the acid improves the situation somewhat but most of the inhibition comes from the chaotropic anion of the acid.
The situation described above has led to a continuous search for alternative extraction methods. Among alternative extractants the quaternary ammonium compounds, e.g. benzalkonium chloride, have been suggested (S. .ANG.nschn, A. Lundin, L. Nilsson and A. Thore, Detection of bacteriuria by a simplified luciferase assay of ATP. Proceedings: International Symposium on Analytical Applications of Bloluminescence and Chemiluminescence, pp. 438-445, State Printing & Publishing, Inc., Westlake Village, Cal., 1979). However, quaternary ammonium compounds inactivate firefly luciferase to give a gradual decay of the light emission after addition of the extract to the firefly reagent. Such a gradual decrease of the luciferase activity during the light measurement makes it almost impossible to calibrate the assay by adding a known amount of ATP (the internal standard technique). In the above paper it was stated that the inactivation effect could be partially counteracted by addition of bovine serum albumin. However, in later efforts to optimise this procedure it was found that the concentrations of albumin needed (2.5-10%) to completely avoid the inactivation of luciferase by quaternary ammonium compounds resulted in a strong inhibition of the luciferase reaction (A. Lundin, Extraction and automatic luminometric assay of ATP, ADP and AMP. In Analytical Applications of Bioluminescence and Chemiluminescence, L. Kricka, P. Stanley, G. Thorpe and T. Whitehead, Eds., pp. 545-552, Academic Press, New York, 1984). The important finding was, however, that the luciferase inactivating effect of quaternary ammonium compounds could be neutralised although albumin was not ideal for the purpose. An alternative neutralising agent for quaternary ammonium compounds 10 was later found to be nonionic surfactants, e.g. Tween 20, Tween 60, Tween 80, Polyoxyethylene ether W1 and Triton X-100 (W. J. Simpson and J. R. M. Hammond, EP 309184). S. Kolehmainen and V. Tarkkanen have proposed (GB 16004249) the use of nonionic surfactants as extractants in their own right. Nonionic surfactants counteract the gradual inactivation of luciferase by quaternary ammonium compounds and are not by themselves strongly inhibitory in the luciferase reaction. However, a considerable inhibition of the luciferase reaction is obtained at the addition of quaternary ammonium compounds even in the presence of nonionic surfactants (cf. Example 1). Thus no system has been described that obviates both problems with quaternary ammonium compounds, i.e. inhibition and inactivation of luciferase.
Considerations underlying the invention as applied to ATP
Transport of samples to a laboratory obviates the major advantage of the firefly ATP assay in rapid microbiology, i.e. the fact that analytical results are provided within minutes. A major potential market for such assays is actually field testing under nonlaboratory conditions using personnel with little or no training in biochemical analysis. Under such conditions assays would normally involve low numbers of samples in each series and would have to be performed with reagents stored at ambient temperature and with low-price and simple instrumentation. Analytical procedures would have to involve a minimum number of very simple steps using reagents-with a-format-suitable for single assays. Prototype analytical systems for such assays based on dipstick technology have been described (A. Lundin, ATP assays in routine microbiology: From visions to realities in the 1980s, in ATP Luminescence: Rapid Methods in Microbiology, P. E. Stanley, B. J. McCarthy and R. Smither, Eds., The Society for Applied Bacteriology Technical Series 26, Blackwell Scientific Publications, pp. 11-30, Oxford, 1989).
A serious problem in the development of commercial reagent kits for the firefly assay would be that the ATP standard is less stable than the firefly luciferin-luciferase reagent. It is unlikely that an ATP standard can be stored for prolonged times at ambient temperatures can be developed. Reconstitution and dispensing of the ATP standard in the assay represent further problems. An ATP standard solution would have to be added in an accurate volume .ltoreq.1% of the total assay volume (A. Lundin, Clinical Applications of Luminometric ATP monitoring, Thesis from Karolinska Institute, 1990). Accurate pipetting of microlitre volumes by untrained personnel under field testing conditions would be very difficult to achieve. The price of automatic equipment would be prohibitive in this market. Even if all the above problems could be solved the internal standard technique makes it necessary to perform two light measurements, i.e. before and after addition of the ATP standard. Thus from several points of view it would be highly advantageous if the assay could be performed without the use of ATP standards. This could be achieved using standardised firefly reagents with an essential stable light emission always having the same relation to the ATP concentration in all samples. Lyophilised firefly reagents that can be stored for years with no loss of activity having an essentially stable light emission during several minutes have been commercially available since the late 1970s (A. Lundin, Clinical Applications of Luminometric ATP Monitoring, Thesis from the Karolinska Institute, 1990). Systems for simple automatic calibration of the light response of light measuring instruments also represent well established technology. The only remaining problem would be to assure that addition of extracts of biological material affects the luciferase activity neither by inactivation (resulting in a decay of the light emission) nor inhibition (resulting in a decreased but stable light emission) during the light measurement.
Very potent extractants that rapidly penetrate the cell wall and inactivate the 20 intracellular enzymes have to be used with microbial cells. The interference with enzymatic analysis from such extractants can be obviated by: 1) Dilution of extracts (resulting in a reduced sensitivity of the assay). 2) Removal of the extractant from the extract (most likely resulting in time-consuming and laborious procedures). 3) Neutralisation of the extractant by including a neutralising agent in the assay buffer. The last suggestion is obviously the most attractive alternative. The requirement for very potent extractants also makes it difficult to achieve. The situation is not simplified by the fact that the neutraliser has to be relatively inert with no effects on luciferase activity.
The overall aim of this aspect of the present invention can be stated as the development of a combination of extractants and neutralisers that causes neither inactivation of luciferase nor inhibition of the luciferase reaction. Only by achieving both these goals convenient and reliable ATP assays can be performed under field testing conditions, i.e. without using ATP standards.
Neutralisation of an extractant can be achieved by performing a chemical reaction to destroy the extractant. The simplest example would be the neutralisation of an acid extractant by addition of a base. However, an exact pH adjustment would be required (strong buffers are inhibitory) and would not be practicable in many situations. Furthermore the best acid extractants have chaotropic anions, which are strongly inhibitory even at neutral pH. Even an increased ionic strength reduces luciferase activity. An alternative approach would be to destroy the extractant by forming a new non-inhibitory compound by a chemical reaction. However, this would most likely have to involve highly reactive reactants that would be likely to inhibit or inactivate enzymes.
The most attractive approach would be to form a complex between the extracting molecule and a neutralising molecule. The use of nonionic surfactants to neutralise quaternary ammonium compounds (a type of cationic surfactants) is an example of this approach (W. J. Simpson and J. R. M. Hammond, European Patent Application 88308677.9). Actually nonionic surfactants neutralise the inactivation effect on firefly luciferase of all types of ionic surfactants (cationic, anionic and zwitterionic) as shown in Example 1. However, in the presence of nonionic surfactants all the ionic surfactants give an inhibitory effect at much lower concentrations than those causing inactivation. This may be due to a poor association between nonionic and ionic surfactants or to an inhibition from the complex between the two types of surfactants. Regardless of explanation the inhibition is likely to vary from sample to sample depending on the level of biological material that may bind extractants of the ionic surfactant type. Thus it would be necessary to use ATP standards in each assay. A further disadvantage of nonionic surfactants as neutralisers is that not all enzymes are as resistant as firefly luciferase to these agents.
The ideal compound for neutralising extractants would have a high association constant for the extractant. Ideally it would form an inclusion complex so that the part of the extractant molecule that inactivates enzymes is surrounded by a protective layer. Obviously the neutralising compound should be as inert with enzymes as possible and should not irreversibly bind intracellular metabolites that are of analytical interest. Some surfactants, e.g. the quaternary ammonium compounds, have been found to be useful extractants (A. Lundin, Extraction and automatic luminometric assay of ATP, ADP and AMP). In Analytical Applications of Bioluminescence and Chemiluminescence, L. Kricka, P. Stanley, G. Thorpe and T. Whitehead, Eds., pp 545-552, Academic Press, New York, 1984). A common feature of all surfactant molecules is a hydrophobic tail. The formation of an inclusion complex in which the hydrophobic tail is buried in a complex with a hydrophilic outer surface would be ideal. This might be achieved using a neutralising agent forming micelies. However, enzymes added in the analytical procedure may become incorporated into the micelles resulting in a changed activity. Furthermore an interaction between the enzymes and the extractants within the micelle can not be excluded. The idea1 neutralising agent for surfactants would be a water-soluble compound with a hydrophilic outer surface not likely to bind to enzymes and a hydrophobic cave with an appropriate size to form inclusion compounds with surfactants.
properties of cyclodextrins
Cyclodextrins are doughnut-shaped molecules consisting of 6, 7 or 8 glucose units (.alpha.-, .beta. and y-cyclodextrin). The internal diameter of the ring is 6 .ANG., 7.5 .ANG.and 9.5 .ANG., respectively. The interior of the ring binds the hydrophobic tails of molecules as e.g. surfactants. The resulting inclusion complexes are generally formed with a 1:1 stoichiometry between surfactant and cyclodextrin. the association constants with .alpha.-, .beta. and y-cyclodextrin depend on the size and chemical properties of the hydrophobic tail of the surfactant. The association constant with surfactants is generally in the range 10.sup.3 -10.sup.4 but may be as high as 5.times.10.sup.4 dm.sup.3 mol.sup.-1 (I. Satake, T. Ikenoue, T. Takeshita, K. Hayakawa and T. Maeda, Conductometric and potentiometric studies of the association of .alpha.-cyclodextrin with ionic surfactants and their homologs, Bull. Chem. Soc. Jpn. 58, 2746-2750, 1985; R. Palepu and J. E. Rickardson, Binding constants of .beta.-pcyclodextrin/surfactant inclusion by conductivity measurements, Langmuir 5, 218-221, 1989; I. Satake, S. Yoshida, K. Hayakawa, T. Maeda and Y. Kusumoto. Conductometric determination of the association constants of .beta.-cyclodextrain with amphiphilicions, Bull. Chem. Soc. Jpn. 59, 3991-3993, 1986; T. Okubu, Y. Maeda and H. Kitano, Inclusion process of ionic detergents with cyclodextrins as studied by the conductance stopped-flow method, J. Phys. Chem. 93, 3721-3723, 1989; R. Palepu and V. C. Reinsborough, Surfactant-cyclodextrin interactions by conductance measurements, Can. J. Chem. 66, 325-328, 1988). The outer surface of the cyclodextrins is hydrophilic and is unlikely to interact with most enzymes. Furthermore the cyclodextrins are water soluble, although they can be immobilised, e.g. by polymerisation or by attachment to a solid or particulate surface. The use of cyclodextrins to remove surfactants from surfaces and solutions have been described. (P. Khanna and R. Dworschack, European Patent Application EP 301,847). According to this patent application surfactants can be removed from solutions by immobilised cyclodextrins. The possibility not to remove but to neutralise the effect of the surfactants by forming inclusion complexes was not evaluated. P. Khanna et al. EP 286367 describe the use of cyclodextrins to neutralise surfactants used as stabilisers of peptide fragments prior to assay. In a review various applications of cyclodextrins in diagnostics have been described (J. Szejtli, Cyclodextrins in diagnostics, Kontakte (Darmstadt) 1988 (1), 31-36). The use of cyclodextrins to neutralise surfactants added as extractants to release lntracellular metabolites has not been previously described.